Method of inspecting staphylococcus aureus

ABSTRACT

This invention relates to a method for testing  S. aureus  in a specimen wherein DNase produced by the  S. aureus  is directly detected or quantitatively determined by an immunoassay without cultivating the  Staphylococcus aureus,  and a test kit for testing  S. aureus  in a specimen by such test method.  
     Use the present invention enables detection of  S. aureus  in the sample in a short time and at a high sensitivity without cultivating the  S. aureus.

TECHNICAL FIELD

This invention relates to a method for directly testing Staphylococcusaureus in a specimen without cultivating Staphylococcus aureus.

BACKGROUND ART

Staphylococcus aureus (S. aureus) is a bacterium of Staphylococcus spp.which is gram-positive and which usually appears under the microscope asirregular grape-like clusters, and it is also a major pathogenicbacterium which produces various toxins and enzymes that induce purulentdiseases in human and mammals. S. aureusis also a typical bacterialstrain which causes food poisoning in human.

A popular procedure that has often been used in detecting S. aureus is aprocedure wherein pure cultivation is conducted in a selective isolationmedium, and suspicious colonies are chosen for further identificationtests of the bacterial species, and the identification has beenconducted by using coagulase produced by S. aureus. Coagulase is anenzyme which coagulates human or rabbit plasma, and S. aureus can bedifferentiated from coagulase-negative staphylococci (CNS) by checkingthe coagulase production.

In addition, DNase test is also used for differentiation of S. aureusfrom CNS since DNase is produced by S. aureus but not by CNS next tocoagulase. In this DNase test, S. aureus is identified by using thephenomenon that, when a streaked DNA plate culture is cultivatedovernight and to which 1.5N hydrochloric acid is added, white cloudingoccurs in the part where DNA is present while such whitening is notobserved near the bacterial plaque where DNA decomposition by the DNasehas taken place.

The conventional test methods, however, all required cultivation of thebacteria, and a substantial time was needed before the identification ofthe bacterium. When the results of the identification were obtained,patients were often too serious to utilize the test results. Inaddition, there has been a fair possibility that the bacterium presentin a minute amount in blood is left undetected in patients receiving alarge amount of antibacterial. Furthermore, the former method wasassociated with the risk that bacteria other than Staphylococcus spp.may grow under the same cultivation conditions.

A method has also been developed in which detection time has beenreduced and specific detection of S. aureus has been enabled. In thismethod, precultivation is conducted and the DNase in the culturesupernatant is measured by an enzyme immunoassay for identification ofthe bacterium (Brakstad et al., APIMS 97, pages 166-174 (1989); Brakstadet al., APIMS 103, pages 209-218 (1995)). Although pure culture is notrequired in this method for identification of the bacterium, aprecultivation is still required to improve the detection sensitivity(detection limit, 0.4 ng/mL or higher). An enzyme immunoassay byfluorescence method has also been developed (Meyrand et al., Lett ApplMicrobiol 29, pages 216-220 (1999)). The detection limit of this method,however, is 1 ng/mL.

Another attempt that has been made for quick, high sensitivity detectionof bacterial infection is gene amplification (Kato, I., Protein, NucleicAcid and Enzyme, Vol. 35, page 2957 (1990)). Since S. aureus is anindigenous bacterium, its detection is associated with the risk sharedby indigenous bacteria that those not infected are also detected aspositive by the minute contamination of the bacterial gene.

In the meanwhile, MRSA (methicillin-resistant S. aureus) has attractedspecial attention among S. aureus since it is a causative bacterium ofnosocomial infection, and a convenient and fast identification method isimportant. MRSA has been traditionally identified by evaluating chemicalsensitivity in a test cultivation of the bacteria separated from aclinical material. However, a method utilizing detection of mecA whichis a gene involved in the antibiotic resistance of MRSA has beenrecently developed, and this method has enabled the MRSA identificationwith no need to wait the results of the sensitivity test by utilizinggene amplification (Ubukata et al., J. Clin. Microbiol., 30, pages1728-1733 (1992)). This method is already in clinical use.

This mecA gene, however, is sometimes found in Staphylococcus other thanS. aureus such as CNS, and detection of mecA gene has been associatedwith the difficulty of clearly differentiating between MRSA and CNS.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a method which iscapable of detecting S. aureus in a specimen in a short time and at ahigh sensitivity without cultivating S. aureus.

In view of the situation as described above, the inventors of thepresent invention have made an intensive investigation on quick testsfor detecting S. aureus in a specimen, and found that an immunoassay ofhigh sensitivity and high precision using an antibody against DNasespecifically produced by S. aureus is capable of directly detecting orquantitatively determining the DNase antigen in the blood, urine,sputum, spinal fluid, pleural effusion, ascites, pus, or other bodyfluid components from patients infected by S. aureus without any needfor cultivating the bacterium, and that S. aureus in a specimen can betested in a short time by using such method. The present invention hasbeen completed on such a finding.

Accordingly, the present invention provides a method for testing S.aureus in a specimen wherein DNase produced by the S. aureus is directlydetected or quantitatively determined by an immunoassay withoutcultivating S. aureus.

The present invention also provides a test kit for testing S. aureus ina specimen by the test method as described above wherein the kit atleast comprises an antibody for the DNase and a reagent for detectingthe labeled DNase.

The present invention has enabled to test S. aureus in a body fluidcomponent without any need for cultivating the S. aureus, and toconveniently and quickly test whether or not the patient is infected byS. aureus. The present invention can also determine the infection by S.aureus including MRSA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view showing the standard curve of purified DNase measuredby sandwich enzyme immunoassay 1.

FIG. 2 is a view showing the standard curve of the purified DNasemeasured by sandwich enzyme immunoassay 2.

FIG. 3 is a view showing the standard curve of the purified DNasemeasured by sandwich enzyme immunoassay 3.

FIG. 4 is a view showing amount of the DNase in the supernatant that hasbeen measured over time.

FIG. 5 is a view showing measurements of the DNase in serum of healthydonors (3 specimens), and serum (8 specimens) and urine (4 specimens) ofthe patients suffering from S. aureus.

BEST MODE FOR CARRYING OUT THE INVENTION

The method for testing S. aureus of the present invention is a methodwherein DNase produced by S. aureus is directly detected in the specimenby an immunoassay.

Accordingly, the present invention does not require precultivation ofthe specimen (test specimen) or increase in the concentration of theDNase in the specimen by centrifugation, column separation,precipitation, or the like.

In the present invention, the term “S. aureus” includsmethicillin-resistant S. aureus.

The specimen used in the present invention is not limited as long asthere is possibility that S. aureus is present, and exemplary specimensinclude blood, urine, sputum, spinal fluid, pleural effusion, ascites,pus, and other body fluid components. More specifically, the presentinvention is capable of directly detecting or quantitatively determiningthe DNase without precultivation of the test specimen or increase in theconcentration of the DNase of the test specimen not only when thespecimen are those containing the bacteria at a high concentration suchas sputum or urine but also when the specimen are those containing thebacteria at a low concentration (for example, 10 to 10³ cfu/mL) such asblood, pleural effusion, or ascites.

In addition to the use of the specimen with no further dilution, thespecimen may also be used after diluting with a buffer such as phosphatebuffer. The buffer used in the dilution of the specimen maysimultaneously contain an adequate protein such as BSA, HSA, gelatin, orthe like for stabilization of the antigen.

The immunoassay used in the present invention is not particularlylimited as long as it is an immunoassay of high sensitivity which canmeasure the DNase produced by S. aureus to the order of 100 pg/mL orless, and more preferably 10 pg/mL or less, and exemplary immunoassaysinclude enzyme immunoassay, radioimmmunoassay, immunochromatography,fluorescent immunoassay, and other immunoassays known in the art. Thepreferred, however, is an enzyme immunoassay in view of the safety andthe sensitivity.

An enzyme immunoassay is an immunoassay wherein amount of the relevantantibody or antigen is measured by detecting enzyme activity of theenzyme labeled antibody using an enzyme such as peroxidase, alkalinephosphatase, or β-galactosidase. Enzyme immunoassays using variousenzyme detection means are known such as colorimetric enzymeimmunoassay, fluorescent enzyme immunoassay, and chemiluminescent enzymeimmunoassay (CLEIA). In the colorimetric enzyme immunoassay, enzymeactivity is quantitatively determined by measuring coloring of thechromogenic substance generated by decomposition of the enzymaticsubstrate such as hydrogen peroxide/o-phenylenediamine,p-nitrophenylphosphate, or o-nitrophenyl-β-D-galactopyranoside by theenzyme. In the fluorescent enzyme immunoassay, fluorescence is generatedby decomposition of the fluorescent substrate such as 4-hydroxyphenylacetic acid, 4-methyl-umbelliferyl phosphate, or4-methyl-umbelliferyl-β-galactoside by the catalytic activity of theenzyme, and the fluorescence intensity is measured to quantitativelydetermine the enzyme activity. In the chemiluminescent enzymeimmunoassay (CLEIA), chemiluminescent substance is excited by thecatalytic activity of the enzyme, and amount of the light emitted whenit returns to the ground state is measured to quantitatively determinethe enzyme activity. Among these, use of a chemiluminescent enzymeimmunoassay is particularly preferable in the present invention since nolight source is required for the chemiluminescence of thechemiluminescent substance, and detection at a sensitivity higher thanthe colorimetric or the fluorescent immunoassays is possible.

Exemplary enzymes used in the CLEIA include peroxidase, alkalinephosphatase, β-galactosidase, glucose oxidase, andglucose-6-phosphodehydrogenase, and exemplary chemiluminescentsubstances include luminols such as luminol derivatives and isoluminolderivatives, lucigenin(N,N′-dimethyl-9,9′-bisacridinium nitrate), andbis(2,4,6-trichlorophenyl)oxalate. Among these, the preferred is thecombination of luminol/hydrogen peroxide for the substrate withperoxidase (Tsuji, A., Protein, Nucleic Acid and Enzyme, SpecialEdition, vol. 31, pages 51-63 (1987)). If necessary, D-(−)-luciferin,6-hydroxybenzothiazole, p-iodophenol, or the like may be simultaneouslyincorporated in the chemiluminescent system to enhance thechemiluminescence.

The immunoassay of the present invention is not particularly limited forits measurement procedure, and the assay may be conducted either by acompetitive assay or a sandwich assay. However, use of a sandwich assayis preferable in view of conducing the assay at a high sensitivity.

Next, an embodiment of the test method for S. aureus using sandwichassay is described in detail.

1) Antibody against DNase produced by S. aureus is immobilized on anadequate solid carrier by adsorption.

2) The antibody is brought in contact with the specimen containing theS. aureus for specific bonding of the DNase produced by the S. aureus inthe specimen with the anti-DNase antibody immobilized on the solidcarrier to thereby immobilize the DNase in the specimen to the solidcarrier.

3) The carrier having DNase immobilized thereon is brought in contactwith a solution containing a labeled antibody against the DNase(secondary antibody) to bind the secondary antibody to the solid carrierthrough the immobilized anti-DNase antibody and the DNase.

4) The thus immobilized secondary antibody is measured by means of thelabel of the secondary antibody to detect or quantitatively determinethe DNase in the specimen.

The antibody against the DNase used in the test method of the presentinvention may be either a polyclonal antibody or a monoclonal antibody,and this antibody can be produced by the method known in the art usingthe purified antigen.

The secondary antibody may comprise an antibody in its entirety.However, in view of reducing the nonspecific reactions to the lowestpossible level, the secondary antibody may preferably comprise afragment of the antibody such as Fab, Fab′, or F(ab′)2 produced bytreating the antibody with an enzyme such as pepsin or by reductiontreatment. The combination of the primary antibody and the secondaryantibody may be a combination of a monoclonal antibody or a fragmentthereof recognizing different epitopes, or a combination of a polyclonalantibody and a monoclonal antibody or a fragment thereof.

Non-limiting Examples of the solid carrier used for immobilizing theprimary antibody include glass or plastic tube, plate, well, or beads,magnetic particles, latex, membrane, and fibers.

The immobilization can be accomplished by physical adsorption, oralternatively, by chemical bonding using a crosslinking agent such asglutaraldehyde or carbodiimide. For example, immobilization may beconducted by contacting the antibody diluted with phosphate buffer withthe surface of the solid carrier, and incubating the carrier at 37° C.for 60 minutes.

The labeling of the secondary antibody may be accomplished by directlabeling with a radioisotope, an enzyme, biotin, a fluorescentsubstance, a chemiluminescent substance, gold colloid, latex, ferriteparticle, or the like. The secondary antibody, however, is preferablylabeled with an enzyme as described above in view of high safety andexpectation for good measurement results. Exemplary preferable enzymesused for the labeling include peroxidase, alkaline phosphatase, andgalactosidase which are highly stable and whose enzyme activity can bereadily determined.

The secondary antibody bonded can be detected or quantitativelydetermined by any method known in the art, for example, by directlydetecting or quantitatively determining the secondary antibody itselfthat has been labeled with an enzyme, a luminescent substance, afluorescent substance, or the like, or by using a tertiary antibodywhich specifically binds to the secondary antibody and labeling thistertiary antibody by various methods and detecting or quantitativelydetermining the label of the tertiary antibody.

The secondary antibody is preferably detected by reacting the secondaryantibody labeled with an enzyme with a substrate (chromogenic substrate,fluorescent substrate, or chemiluminescent substrate) which is specificfor the enzyme, and detecting the signal associated with the colordevelopment, fluorescence, luminescence, or the like induced by thereaction with a measuring device. Use of the chemiluminescent substratewhich enables high sensitivity detection is particularly preferable.

The test kit for infections caused by S. aureus of the present inventionis a kit for carrying out the test method for S. aureus in the specimenas described above, and the kit at least comprises an antibody againstthe DNase as described above (two antibodies when the immunoassay usedis a sandwich assay), and a reagent for detecting the label such as anenzyme, and optionally, a buffer solution for diluting the specimen, abuffer solution for diluting various reagents, a washing solution, andthe like.

EXAMPLES

Next, the present invention is described in further detail by referringto the Examples which by no means limit the scope of the invention.

Example 1 (1) Preparation of a Plate Having Primary Antibody ImmobilizedThereto

Rabbit or sheep anti-DNase polyclonal antibody diluted with 25 mMphosphate buffer solution (PBS) containing 150 mM NaCl to 2 μg/mL wasdispensed in the wells of a plate at 50 μL/well, and the plate wasallowed to stand overnight at 4° C. for immobilization of the antibody.The content was removed from the plate the next day, and after adding280 μL/well of PBS containing 0.2% bovine serum albumin (BSA) (0.2%BSA-PBS), the plate was allowed to stand overnight or longer at 4° C. Inuse, the plate washed three times with PBS containing 0.05% Tween20(PBS-T) was used for the reaction.

(2) Preparation of Biotinylated Secondary Antibody

500 μL (1 mg/mL) of sheep anti-DNase polyclonal antibody was dialyzedagainst 0.1M sodium hydrogen carbonate. A solution of 0.05 mg ofSulfo-NHS-LC-Biotin (manufactured by Pierce) in 50 μL of the carbonatebuffer solution was added dropwise to the antibody solution withstirring. After stirring at room temperature for 4 hours, and dialyzingagainst PBS overnight at 4° C., the antibody solution was used as thebiotinylated secondary antibody.

Example 2 (1) Measurement of DNase by Sandwich-Enzyme Immunoassay (1)

50 μL/well of purified DNase (manufactured by Toxin Technology) dilutedwith 0.2% BSA-PBS to 0.1, 1, 10, and 100 ng/mL was added to the wells ofthe microtiter plate having the sheep anti-DNase polyclonal antibodyimmobilized thereto produced in Example 1(1). After allowing thereaction to take place at room temperature for 1 hour, the wells werewashed with PBS-T, and 50 μL/well of rabbit anti-DNase polyclonalantibody diluted with 0.2% BSA-PBS to 2 μg/mL was added as the secondaryantibody. The reaction was then allowed to proceed at room temperaturefor 1 hour, and the wells were washed with PBS-T. In the meanwhile,HRP-labeled pig anti-rabbit IgG antibody (manufactured by DAKO) wasdiluted with 0.2% BSA-PBS to 4000 folds, and 50 μL/well of this dilutionwas added to the well, and the reaction was allowed to proceed at roomtemperature for 1 hour. After washing, 50 μL/well of 0.1M citrate buffersolution containing 2 mg/mL o-phenylenediamine and 9.5 mM hydrogenperoxide, pH 5 was added, and the reaction was allowed to proceed atroom temperature for 30 minutes, and the reaction was ceased by adding50 μL/well of 1.5N sulfuric acid. The measurement was conducted by usinga plate reader for ELISA at a wavelength of 492 nm.

The resulting standard curve is shown in FIG. 1. The measurement waspossible in the DNase concentration range of 0.1 to 100 ng/mL.

(2) Measurement of DNase by Sandwich Enzyme Immunoassay (2)

50 μL/well of purified DNase diluted with 0.2% BSA-PBS to 0.1, 1, 10,100, and 1000 ng/mL was added to the wells of the microtiter platehaving the rabbit anti-DNase polyclonal antibody immobilized theretoproduced in Example 1(1). After allowing the reaction to take place atroom temperature for 1 hour, the wells were washed with PBS-T, and 50gL/well of biotinylated sheep anti-DNase polyclonal antibody dilutedwith 0.2% BSA-PBS to 3 μg/mL produced in Example 1(2) was added as thesecondary antibody. The reaction was then allowed to proceed at roomtemperature for 1 hour, and the wells were washed with PBS-T. In themeanwhile, avidinylated HRP (manufactured by Zymed) was diluted with0.2% BSA-PBS to 4000 folds, and 50 μL/well of this dilution was added tothe well, and the reaction was allowed to proceed at room temperaturefor 1 hour. After washing, 50 μL/well of 0.1M citrate buffer solution,containing 2 mg/mL o-phenylenediamine and 5.9 mM hydrogen peroxide, pH 5was added, and the reaction was allowed to proceed at room temperaturefor 30 minutes, and the reaction was ceased by adding 50 μL/well of 1.5Nsulfuric acid. The measurement was conducted by using a plate reader forELISA at a wavelength of 492 nm.

The resulting standard curve is shown in FIG. 2. The measurement waspossible in the DNase concentration range of 0.1 to 100 ng/mL.

(3) Measurement of DNase by Sandwich Enzyme Immunoassay (3)

50 μL/well of purified DNase diluted with 0.2% BSA-PBS to 1, 10, 100,and 1000 pg/mL was added to the wells of the white microtiter plate(manufactured by Dynex technologies) having the rabbit anti-DNasepolyclonal antibody immobilized thereto. After allowing the reaction totake place at room temperature for 1 hour, the wells were washed withPBS-T, and 50 μL/well of biotinylated sheep anti-DNase polyclonalantibody diluted with 0.2% BSA-PBS to 3 g/mL produced in Example 1(2)was added as the secondary antibody. The reaction was then allowed toproceed at room temperature for 1 hour, and the wells were washed withPBS-T. In the meanwhile, avidinylated HRP was diluted with 0.2% BSA-PBSto 4000 folds, and 50 μL/well of this dilution was added to the well,and the reaction was allowed to proceed at room temperature for 1 hour.After washing, 100 μL/well of SuperSignal ELISA Femto Substrate(manufactured by Pierce) was added, and luminescence of 10 seconds wasintegrated with a luminometer.

The resulting standard curve is shown in FIG. 3. The measurement waspossible in the DNase concentration range of 0.1 to 1000 μg/mL.

Example 3 Measurement of the Amount of DNase in the Culture SupernatantOver Time

To 10 mL of heart-infusion broth was added 80 μL of overnight culture ofthe standard strain of S. aureus (NCTC8325), and the cultivation wascontinued. 1 mL portion of the culture was collected over time, and thecollected culture was centrifuged for 2 minutes. The supernatant wasfiltered with 0.2 μm membrane, and the filtrate was stored at −30° C.until measurement. In order to measure the amount of the DNase secretedin accordance with the growth of the bacterium, the sample was diluted100 to 5000 folds with 0.2% BSA-PBS for measurement by ELISA.

The results are shown in FIG. 4. As demonstrated by the results,production of DNase increased corresponding to the growth phase of thebacterium.

Example 4 Measurement of DNase in the Serum and Urine of the Patients

DNase in the serum and urine of the patients suffering from S. aureusand the healthy donors were measured according to the proceduredescribed in Example 2 (3). The results are shown in FIG. 5. While noDNase was detected in the serum of the healthy donor, presence of DNasewas confirmed in the serum and the urine of the infected patients.

1. A method for testing Staphylococcus aureus in a specimen whereinDNase produced by the Staphylococcus aureus is directly detected orquantitatively determined by an immunoassay without cultivating theStaphylococcus aureus.
 2. The test method according to claim 1 whereinthe immunoassay is an enzyme immunoassay.
 3. The test method accordingto claim 1 wherein the immunoassay is an enzyme chemiluminescentimmunoassay.
 4. The test method according to claim 3 wherein the enzymechemiluminescent immunoassay is the one using peroxidase for the enzymeto be labeled and a luminol for the chemiluminescent substance.
 5. Thetest method according to any one of claims 1 to 4 wherein the specimenis a body fluid component.
 6. The test method according to anyone ofclaims 1 to 5 wherein Staphylococcus aureus is methicillin-resistantStaphylococcus aureus.
 7. A test kit for testing Staphylococcus aureusin a specimen by the test method according to any one of claims 1 to 6wherein the kit at least comprises an antibody for the DNase and areagent for detecting the labeled DNase.